RESUMO
Orthodontic tooth movement elicits alveolar bone remodeling and orofacial pain that is manifested by tooth mechanical hyperalgesia. Nerve growth factor (NGF) is upregulated in periodontium and may modulate tooth mechanical hyperalgesia. The objectives were to examine the role of NGF in tooth mechanical hyperalgesia and to elucidate the underlying mechanisms. Tooth mechanical hyperalgesia was induced by ligating closed coil springs between incisors and molars in Sprague-Dawley rats. Retrograde labeling was performed by periodontal administration of fluor-conjugated NGF and the detection of fluorescence in trigeminal ganglia (TG). Lentivirus vectors carrying NGF shRNA were employed to knockdown the expression of NGF in TG. The administration of agonists, antagonists, and virus vectors into TG and periodontium was conducted. Tooth mechanical hyperalgesia was examined through the threshold of biting withdrawal. Our results revealed that tooth movement elicited tooth mechanical hyperalgesia that could be alleviated by NGF neutralizing antibody and that NGF was upregulated in periodontium (mainly in periodontal fibroblasts) and TG. Retrograde labeling revealed that periodontal NGF was retrogradely transported to TG after day 1. Acid-sensing ion channel 3 (ASIC3) and NGF were co-expressed in trigeminal neurons and the percentage of co-expression was significantly higher following tooth movement. The administration of NGF and NGF neutralizing antibody into TG could upregulate and downregulate the expression of ASIC3 in TG, respectively. NGF aggravated tooth mechanical hyperalgesia that could be alleviated by ASIC3 antagonist (APETx2). Moreover, NGF neutralizing antibody mitigated tooth mechanical hyperalgesia that could be recapitulated by ASIC3 agonist (GMQ). NGF-based gene therapy abolished tooth mechanical hyperalgesia and downregulated ASIC3 expression. Taken together, in response to force stimuli, periodontal fibroblasts upregulated the expressions of NGF that was retrogradely transported to TG, where NGF elicited tooth mechanical hyperalgesia through upregulating ASIC3. NGF-based gene therapy is a viable method in alleviating tooth-movement-induced mechanical hyperalgesia.
RESUMO
Objective:]To investigate the role of Arpin protein in bone repair by mediating migration of host bone marrow mesenchymal stem cells (BMSCs) to the bone defect area after transplantation of tissue engineering bone (TEB).Methods:Immunofluorescence was used to observe the expression and relative localization of Arpin and Arp2/3 proteins in BMSCs. Lentiviruses that ware designed to interfere with Arpin expression were constructed to transfect BMSCs for knockdown Arpin expression. Knockdown efficiency was verified by real-time quantitative reverse transcription PCR ( qRT-PCR) and Western blot. According to different levels of Arpin protein expression, experiments were divided into empty vector control group and an Arpin expression inhibition group in vitro and in vivo. In vitro experiments: the cell migration model was established with a migration chamber, then the cells from both groups were seeded on the up chamber, and the number of migrated cells were detected by fluorescence microscopy. Cells from both groups were seeded on six-well plates. Model of wound healing experiment was established and wound healing ratio was examined by microscopy. In vivo experiments: 8-week-old C57BL/6 mice were selected and assigned to empty vector control group and Arpin expression inhibition group according to the random number table, with 6 rats per group. Diaphysis of 2 mm and periosteum in the middle femur were excised to make a large segment of bone defects. Then, TEB was transplanted into the defect area and fixed.Green fluorescein-labeled BMSCs (1 million cells per mouse) from empty vector control group and Arpin expression inhibition group were injected through the tail vein. Number of BMSCs homing to the bone defect area was detected by immunofluorescence staining at day 2 and 7 after operation. At 4 weeks after operation, the femur was taken for a Micro-CT scan to analyze bone mass density(BMD), bone volume density (BV/TV), trabecular spacing (Tb.Sp) and trabecular thickness (Tb.Th). Then, the specimens were stained with pathological HE and MASSON staining to observe the quality of bone formation. Results:Mouse BMSCs expressed Arpin protein, which was located at the cell edge relative to Arp2/3. After transfection of lentivirus, BMSCs expressed green fluorescent protein, and the expression of Arpin gene and protein in Arpin expression inhibition group were decreased compared to empty vector control group ( P<0. 01). BMSCs migration was enhanced in Arpin expression inhibition group compared to empty vector control group [(76.6±6.6) vs. (105.7±6.5)] ( P<0. 01). Wound healing was accelerated in Arpin expression inhibition group compared to empty vector control group [(43.8±0.19)% vs. (62.6±3.2)%]( P<0.01). At day 2 after operation, immunofluorescence results showed no significant difference in cell migration between the two groups and almost no labeled cells migrated. At day 7 after operation, more cells migrated to the transplanted area in Arpin expression inhibition group compared to empty vector control group [(5.7±1.5) vs. (11.3±1.5)] ( P<0.01). At 4 weeks after operation, Micro-CT results showed that Arpin expression inhibition group had better bone formation quality than empty vector control group [BMD: (172.7±6.0)mg/cm 3vs. (140.0±6.0)mg/cm 3, BV/TV: (28.8±1.3)% vs. (23.4±0.9)%, Tb.Sp: (0.33±0.01)μm vs. (0.28±0.01)μm, Tb.Th: (0.11±0.01)μm vs.(0.15±0. 01)μm]( P<0.05). Pathological staining showed there were more new bone tissue in Arpin expression inhibition group ( P<0.01). Conclusion:Silencing Arpin protein expression promotes BMSCs to migrate to the bone defect area and improves bone repair effect.
RESUMO
Objective To investigate the mechanism of implanted tissue-engineered bone (TEB)recruiting endogenous mesenchymal stem cells (BMSCs) towards bone regeneration after traumatic bone defect.Methods In vivo experiments:2 mm of diaphysis and periosteum were removed from the middle of the femoral shaft in 8 week old FVB/N mice to form a large segment of bone defect.Demineralized bone matrix (DBM) and TEB were implanted into the defect area and fixated.All mice were randomly divided into DBM group (n =18) and TEB group (n =18).The results were observed 24 hours after implantation:(1) flow cytometry was used to evaluate the number of mobilized host BMSCs into the blood;(2) non-invasive bioluminescent imaging was used to observe the ability of two groups in recruiting mouse bone marrow derived mesenchymal stem cells (mBMSCs) in peripheral blood to the defect area;(3) ELISA was used to evaluate the stromal cell-derived factor 1 (SDF-1) content in peripheral blood of two groups.In vitro experiments:(1) transwell assay was conducted to evaluate the ability of SDF-1 (100 ng/ml) in promoting the migration of human bone marrow derived mesenchymal stem cells (hBMSCs).SDF-1/C-X-C motif chemokine receptor-4 (CXCR4) pathway was blocked by the selective CXCR4 antagonist Plerixafor (AMD3100).The experimental groups were divided into control group,SDF-1 group,and SDF-1 + AMD3100 group.(2) The co-culture system of human umbilical vein endothelial cells (hUVECs) and hBMSCs was established,and cells were stimulated by SDF-1.The experimental groups were divided into hBMSCs group,hBMSCs + hUVECs group,and hBMSCs + hUVECs (AMD3100 pretreatment) group.Transwell assays were used to compare the migration of hBMSCs in each group.ELISA was used to detect the concentration of hepatocyte growth factor (HGF) in the co-culture supernatant.(3) In vitro cultured hUVECs were stimulated by SDF-1 and SDF-1/CXCR4 pathway was antagonized by AMD3100.The experimental groups were divided into control group,SDF-1 group,and SDF-1 + AMD3100 group.Quantitative real-time polymerase chain reaction (qRT PCR) was used to evaluate the expression of HGF in each group.Results In vivo experiments:24 h after transplantation,the number of BMSCs and SDF-1 concentration in the TEB group were significantly highcr than those in the DBM group (P ＜ 0.05).The number of recruited mBMSCs into the circulation in the TEB group was larger than that in the DBM group (P＜ 0.01).In vitro experiments:(1) compared with the control group and the SDF-1 + AMD3100 group,the SDF-1 group significantly enhanced the migration ability of hBMSCs in Transwell migration experiments (P ＜ 0.01);(2) compared with the hBMSCs group and the hBMSCs + hUVECs (AMD3100 pretreatment) group,the number of migrated cells and HGF concentration in the hBMSCs + hUVEC group significantly increased (P ＜ 0.01),but there were no significant differences between the hBMSCs group and the hBMSCs + hUVECs (AMD3100 Pretreatment) group (P ＞0.05);(3) qRT-PCR showed that the expression of HGF was significantly increased in the SDF-1 group compared with the control group (P ＜ 0.05).After antagonizing SDF-1/CXCR4,HGF expression in the SDF-1 + AMD3100 group was significantly lower than that in the SDF-1 group.Conclusions TEB transplantation in traumatic bone defect can significantly increase the concentration of chemokine SDF-1 in vivo and effectively promote the mobilization of endogenous MSCs and recruitment of circulating MSCs.SDF-1 not only directly promotes the migration of hBMSCs through SDF-1/CXCR4 pathway,but also up-regulates the expression and secretion of HGF in vascular cells to further amplify the chemotactic effect of SDF-1 on hBMSCs.